We are combining biochemical and imaging approaches to understand the molecular mechanism of biological motility that depends on actin polymerization. Such motility plays a central role in cell migration, uptake of nutrients by endocytosis, and spreading of pathogens. The pathogenic bacterium Listeria provides a model system for biochemical approaches. By breaking Listeria motility into subreactions, and fractionating brain extracts to identify the proteins required for each, we expect to determine the full biochemical requirements for Listeria motility. We will test the role of new factors identified in this way in three systems: Listeria motility in cells, actin dynamics and endocytosis. We will also determine whether the force for Listeria motility is generated directly by actin polymerization, or by the action of some ATPase motor protein we may identify by fractionation. To probe actin dynamics related to cell migration we will express GFP-tagged actin, and follow its dynamic behavior in the leading edge of spreading cells by fluorescence imaging. We will measure the dynamic behavior of thin spikes (filopodia) and sheets (lamellipodia) at the leading edge, and compare their responses to perturbation of proteins that may be important in generating actin dynamics, notably proteins implicated by our Listeria biochemistry. For perturbation experiments we will use protein tools, and also cell permeable chemicals coming out of a screening effort at the Harvard Institute of Chemistry and Cell Biology (ICCB).